The goal of this research effort is to understand how important types of white blood cell, called T and B lymphocytes, recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognizes a normal component of the body (an "auto-antigen"). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending lymphocytes cells or the auto-reactive cells and how recognition of these antigens is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. Our previous work has described the events within a cell that bring together the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). We are now conducting studies primarily at the cell and tissue level to relate the physiology of antigen recognition to the development of effector function, immune memory, or tolerance. By understanding these events, we will be able to determine how best to deliver antigenic substances to stimulate an effective immune response or to interfere with autoimmune reactions. We previously reported a new method for the direct confocal microscopic visualization in real time of the interactions of T cells and antigen presenting cells in intact lymphoid tissue. These studies showed that individual T cells bind stably to antigen-bearing dendritic cells for several hours before activation-associated events lead to dissociation of the cell pairs and rapid migration of the T cells in the lymphoid tissue. Immunological synapses between T cells and a critical antigen presenting cell (the dendritic cell) were visualized in vivo as was clonal T cell division. This experimental system has now been improved by moving to multiphoton rather than confocal imaging and true intravital observation methods have been developed. We have worked out methods for imaging the bowel, which have allowed us to directly image the interaction of microorganisms with gut epithelium and dendritic cells in the mucosal immune system. Our studies have revealed the dynamic extension of lamina propria dendritic cell processes across the villus epithelium and the capture of lumenal bacteria by these processes. Ongoing studies are examining the movements of B cells in lymphoid tissues and have provided evidence that dendritic cells outside of the follicle are involved in presentation of antigen not only to T cells but also to B cells. We are now able to routinely image peripheral organs and tissues such as the liver, kidney, and skin so that effector functions during infectious and inflammatory processes can be observed. Using these new methods, we are in the midst of a long-term study of lymphoid and myeloid cell dynamics in BCG-induced granulomas in the liver, analyzing the early events following natural bite infection of animals with Leishmania, investigating lymphocyte and DC migration dynamics in peripheral inflamed sites such as the skin, and examining the intersection of the nervous system with the immune and vascular systems. Ongoing work is also aimed at developing tools for in situ imaging of the pulmonary tract. New transgenic mice with cells ubiquitously expressing fluorescent proteins are being generated to provide new tools for long-term tracking of cells and genetically engineered strains of mice that have fluorescent reporters under the control of cytokine gene regulatory elements are being employed to relate the physical behavior of immune cells to their function.